Heterotopic ossification (HO) is a pathologic process resulting in mineralization and potential ossification of soft tissue structures. For reasons largey unknown, HO often occurs following skeletal muscle injury, imposing significant morbidity long after the initial traumatic insult due to pain, muscle loss and restricted muscle/joint motion. Currently, there are no safe pharmacological or surgical means of preventing or selectively removing HO. Thus, there is a need for elucidation of the underlying cause of HO to guide strategic development of novel therapies designed to prevent and treat this devastating disease. Hence, we explored a mechanism suggested by the clinical observation that HO most commonly occurs following injuries that impair the function of the fibrinolytic arm of the coagulation system such as: burns, blast wounds and head trauma. In support of our hypothesis we have discovered that mice with only a partial deficiency of plasminogen (Plg-/+), the precursor of plasmin and primary protease of the fibrinolytic system, develop robust HO in skeletal muscle after injury. In addition, we determined that targeting the primary inhibitor of plasmin, ?2-antiplasmin, prior to skeletal muscle injury in Plg-/+ mice completely prevents HO. Aims: We postulate that 1) a threshold level of plasmin activity is required to prevent initiation f HO after skeletal muscle injury and 2) enhanced plasmin activity can prevent initiation of HO and/or resolve immature HO prior to maturation. Methods: Both aims employ our established Cardiotoxin-induced HO model. Aim 1 will make use of antisense oligonucleotides (ASO) designed to incrementally knock down plasminogen expression in wild type mice prior to HO induction, and disease progression will be monitored with X-ray and microCT. Plasminogen will be measured by gene expression, plasma antigen and plasmin activity levels. In Aim 2 of this proposal we will dose Plg-/+ mice with an ASO targeting ?2-antiplasmin to enhance plasmin's activity either immediately following injury or two weeks after injury, after the development of HO. Plasminogen expression, plasmin activity and development of HO will be monitored as in Aim 1. Impact: The experiments proposed here have the potential to elucidate a mechanism underlying acute HO thereby directly impact clinical treatment of patients with HO. Clinically: Our experiments could help identify patients at risk for HO and improve clinical outcomes, as this work provides necessary information for future studies designed to quantify fibrinolytic activity as it relates to HO in humans. In addition, as soft tissue calcification plays a significat role in multiple diseases, these findings have the potential to reveal novel means of preventing and treating other pathologic processes such as atherosclerosis and valvular calcification. Therapeutically: This work may provide proof-of-principle evidence for a novel pharmacological approach, including a targeted dosing strategy, to prevent, arrest and remove HO through enhanced fibrinolysis.